Information about the identity and total amount of microbes in biological samples is of prime importance in medicine in order to assess the risk of infectious disease, to diagnose infections and predict their clinical course. In a variety of other areas such as food product monitoring, bioremediation, microbial forensics and biowarfare/bioterror investigations, efficient and cost effective methods for quantification of microbial bioagents are needed. In addition, determination of the quantity of a bioagent (microbe, bacterium, virus, fungus, etc.) is a common endeavor in microbiology in the fields of clinical diagnostics, epidemiology, forensics, bioremediation, and quality control.
Methods currently in use for detection and determination of bacteria include bacterial culture and microscopy, detection of bacterial metabolites, and identification of surface molecules by specific antibodies.
The polymerase chain reaction (PCR) is only a qualitative method due to its exponential time course and equally exponential amplification of errors. Efforts have been made to convert PCR to a quantitative method. Among the variety of quantitative PCR methods, are methods depending upon external standardization and on internal standardization. Among the latter, competitive PCR methods are based on co-amplification of a target DNA with a standard competitor DNA which competes with the template DNA for the same set of amplification primers. Since the competitor is added to the PCR reaction mixture in known amounts, it is possible to calculate the amount of target DNA from the experimental determination of the ratio of amplified products of sample and standard competitor DNA.
Methods for rapid and cost effective identification of microbial bioagents through molecular mass measurement of amplification products by molecular mass analysis of bioagent identifying amplicons are disclosed and claimed in U.S. application Ser. Nos. 09/798,007, 09/891,793, 10/660,997, 10/660,122, 10/660,996, 10/418,514 and 10/728,486, each of which is commonly owned and incorporated herein by reference in its entirety. These methods and others would derive great benefit from a means of determination of the quantity of any given microbial bioagent present in a biological sample. Quantification of organisms can be very valuable, particularly in a clinical setting, like Hepatitis C for example, where the greater the number of infectious organisms generally correlates with a less healthy patient and a more difficult clinical course.
The methods described herein satisfy the need for methods for concurrent identification and quantification of bioagents, as well as other needs, by providing internal calibration using a nucleic acid standard calibrant in an amplification reaction.